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As is often the case, an outsider can give fresh perspectives in ways that someone from the inside has difficulty seeing. In this article, Dr. Vincent Marchesi, a former member of the Alzheimer Research Forum Advisory Board, presented a very thoughtful perspective and hypothesis concerning Aβ-mediated toxicity in brain. The premise with which Dr. Marchesi approached this article is that while the evidence linking Aβ peptide to "the pathogenesis of AD is substantial," how these peptides may be toxic in brain is far from resolved. While current investigators favor the model that neurons process APP and release the cleaved Aβ peptides into the extracellular space where they can form cytotoxic oligomers and aggregate into amyloid deposits, this scenario is indeed far from proven. For example, the Golde laboratory recently showed quite definitively that Aβ42 is necessary for parenchymal amyloid deposits when they creatively fused the Aβ domain to the Bri gene and expressed this construct in mice (1). However, these animals, in spite of substantial amyloid load in brain, have a normal lifespan and do not show "obvious behavioral abnormalities." If this is true, then it suggests that lots of extracellular Aβ, whether oligomers or aggregated fibrils, are insufficient to injure neurons or cause synaptic dysfunction.

The central thesis of Dr. Marchesi, then, is that following cleavage of APP by various secretases or other proteases, part or all of the APP transmembrane domain remains within the membrane. The retained membrane segment containing part of the Aβ sequence subsequently interacts with APP and other membrane proteins and, in so doing, perturbs the normal processing of these proteins. Without being specific, Dr. Marchesi speculates that the function of cell surface receptors, ion channels, etc., can be altered by interacting Aβ fragments. The premise behind this novel idea is that both Aβ and APP exist in dimers, presumably within plasma membrane. The APP and Aβ dimers are held together noncovalently through the consensus GxxxG motif that is present in many single transmembrane proteins, including APLP1, Aph-1, and glycophorin A. Indeed, he speculates that APP may be cleaved by secretases as a dimer and the resultant Aβ peptides would remain as dimeric assemblies. The finding that APP dimerization enhances Aβ production is consistent with this view (2). Further, presenilin has been proposed to exist as a dimer in the γ-secretase catalytic core (3). Admittedly, the latter does not mean the APP substrate is also in dimeric form, but then why not? In this model, a portion of Aβ following secretase cleavages remains in, and, in fact, never leaves the membrane. In a postmitotic neuron, Aβ could be stuck in the membrane for a long time, enough to create havoc on neuronal function as it builds up over time.

One can be nitpicky about details of this model, especially where supportive evidence is wanting. But, as a speculative position paper, there is a lot to like about this model. For one, this hypothesis is reminiscent of cytotoxicity mediated by the APP C-terminal fragments first proposed by Neve and Yanker, as well as by Fukuchi and Martin, and others (4,5). These are membrane-retained APP fragments created after a- and especially β-secretase cleavages. While there is no consensus as to how C-terminal APP fragments are toxic, their levels are increased in cells expressing various familial AD-associated APP and presenilin mutations. Furthermore, work from Fred Van Leuven's and Jie Shen's laboratories has shown that loss of PS1 activity in adult brain did not rescue long-term behavioral deficits in the animals even though amyloid production was virtually shut down (6,7). Whether neuronal dysfunction is due to the accumulation of the C99 APP C-terminal fragment is unclear, but is certainly consistent with the putative neurotoxicity of these peptides and quite possibly with Marchesi's model, as well. Second, we and others have proposed a model of cellular toxicity mediated by APP dimerization (8-10). Thus, APP dimers may be bad for the cell by augmenting Aβ production (both released and membrane-retained) and activating signals that can result in neuronal death. Finally, Dr. Marchesi cautions that experimental therapies aimed at reducing soluble Aβ may not be targeting the most appropriate sites. Though speculative, could this be an explanation for the lack of clinical response in the aborted active Aβ vaccination trial from Elan?

In sum, perhaps this idea will be most appealing to those of us who find the myriad of proposed pathways to explain Aβ toxicity rather unsatisfying. Secreted Aβ, whether soluble or oligomeric, could be toxic in vivo, but it should not be so hard to demonstrate this. In the end, maybe this is similar to the story of the drunk looking for his car keys under the street lamp at night. When asked why he was looking there, he replied that he was there because there was light for him to look around even though he dropped his keys somewhere over in the darkened field. So as suggested by Dr. Marchesi, could we have been looking at the wrong place, after all? Or, to paraphrase a now famous political statement: "It's the membrane, stupid."

It is quite surprising that, despite a huge number of experiments done and papers published, the molecular events responsible for the Alzheimer's phenotype remain obscure. This article is a stimulating exercise that gives support to an alternative explanation for the possible toxic effects of Aβ peptides. In a nutshell, Dr. Marchesi proposes that Aβ peptides exert their toxicity within the cell membranes, where they remain entrapped as dimers after the cleavage of APP. Therefore, extracellular oligomers or larger aggregates could have no toxic effects: Plaques could be even a "safe" storage of toxic peptides, which prevent their reassociation with the membranes.

I'll add three further points to the interesting speculation of Marchesi. First, we should consider that Aβ42 has two extra residues (Ile and Ala) at its C-terminus. These could render more stable, compared to Aβ40, the association of the peptide itself with the hydrophobic environment of the membrane bilayer. This could explain why Aβ42 seems to be so crucial for AD pathogenesis.

A second point is that the so-called p3 peptide, always considered not to be toxic, could on the contrary be even more toxic than Aβ. It is generated through the concerted cleavage of APP by a- and ?-secretase, and, as predicted by Marchesi's hypothesis, it could remain inserted in the membrane like Aβ. Furthermore, given that it lacks most of the hydrophilic or charged residues present in the N-terminal half of Aβ, its interaction with the membrane could be more stable than that of Aβ.

Thirdly, we should observe closely the efforts of numerous research groups that are trying to address the possible relationship between lipid rafts, cholesterol content of membranes, ApoE alleles, and AD. Several clinical studies indicate that individuals treated with statins (cholesterol-lowering drugs) show a low incidence of AD (1). These results apparently conflict with the observation that mice treated with statins show a dramatic increase of Aβ production and of amyloid plaques. Marchesi's hypothesis could easily explain this contradiction: The decrease of membrane cholesterol provokes a release of Aβ peptides from the membranes, so that they accumulate in the extracellular space where they are not toxic. In this way, the subjects treated with statins could gain protection from AD while the mice treated with the same drug have more Aβ and more plaques.

I think that this hypothesis deserves to have the field put it to the test with experiments.

In his PNAS Perspective, Vincent Marchesi, in characteristic fashion, provides us with substantial food for thought. Dr. Marchesi presents a cogently argued hypothesis that provides an alternative—or perhaps an addition—to the concept that Aβ oligomers and/or amyloid fibrils injure neurons from without. Based on reported evidence that APP and Aβ can each occur as dimers in cholesterol-rich "lipid raft" domains of neuronal membranes, Dr. Marchesi proposes that APP dimers may be processed as such by the β- and γ-secretase s to yield Aβ dimers which, at least in part, remain in the membrane bilayer. He then suggests specific ways in which intramembranous dimers of Aβ could compromise the function of numerous other transmembrane proteins that share with Aβ the hydrophobic GxxxG motif, including the very proteins that help generate Aβ in the first place (presenilin and Aph-1). If membrane-retained rather than secreted Aβ is principally responsible for compromising neuronal function, Dr. Marchesi notes, perhaps experimental treatments designed to lower extracellular Aβ may miss their mark.

That such a biological scenario could occur seems entirely plausible, and Dr. Marchesi marshals supportive evidence from the literature on the properties of transmembrane segments of proteins, including his own pioneering studies of glycophorin A. But as he also emphasizes, there is as yet no direct experimental evidence that APP dimers are converted per se to Aβ dimers, that Aβ occurs preponderantly as dimers in neuronal membranes, and that these dimers are retained within the membrane for periods sufficiently long to be responsible for triggering the neuronal alterations one observes in Alzheimer disease. This is as it should be for a provocative new hypothesis. I join Drs. Koo and Russo in thanking Dr. Marchesi for creatively pondering the unsolved issue of Aβ's pathogenic mechanism. But I would also like to provide responses to Dr. Marchesi's article that address both certain specific statements he makes and the broader therapeutic implications of his model.

First, Dr. Marchesi states that the findings of LaFerla and colleagues in the triple transgenic mouse model "confirm what many previous investigators have noted, that the initial manifestations of Aβ accumulation begin inside neurons, with the earliest detectable material located inside membranous compartments," including endosomes and lysosomes. In this regard, one needs to distinguish data about the sites of normal Aβ generation throughout life, which include both secretory and endosomal compartments, from data about where Aβ can first be shown to accumulate abnormally during the prodromal stages of AD. As regards the latter, while early intracellular accumulation can be observed in overexpressing disease models such as the triple transgenic mouse (1) and conventional APP transgenic mice (2), it is far more difficult to ascertain the temporal pattern in humans, as we rarely examine the brain tissue before the end of the disease. In the case of human trisomy 21, however, one can detect many diffuse extracellular Aβ deposits in Down subjects as early as 10-12 years of age, and sensitive immunohistochemistry has generally not revealed clear-cut intraneuronal Aβ? aggregates at this early juncture [see, for example, (3)]. Whereas intracellular Aβ can be detected by immunoelectron microscopy in postmortem AD cortex [e.g., (2)], innumerable extracellular deposits are also present. A study of primary neurons cultured from Down syndrome brains observed robust intracellular Aβ staining (4), but this could be due to the increased expression of APP in this particular situation; in conventional AD, wild-type APP is expressed at normal levels. Whereas intracellular tau aggregates (i.e., neurofibrillary tangles as well as "pre-tangle" tau accumulation) are readily detectable by immunohistochemistry in AD and Down postmortem brains, it has been difficult to similarly detect clear-cut intraneuronal Aβ deposits. In our laboratory, Dominic Walsh first detected soluble Aβ dimers and trimers inside certain APP-expressing cultured cells, apparently before they were exported into the medium, leading to our proposal that Aβ dimerization may begin within the vesicles in which it is generated (5). But this does not tell us where Aβ oligomers first accumulate to levels that might actually be injurious to neurons. Therefore, I don't believe that existing evidence supports Dr. Marchesi's rather definitive statement that I quote above. In this sense, I don't concur with Dr. Marchesi's inference that "Aβ peptides do eventually accumulate outside cells as the disease progresses." Rather, they can accumulate outside cells early on (long before clinical symptoms), and longitudinal studies of Down syndrome brains of increasing age as well as APP transgenic mouse brains support this conclusion.

Dr. Marchesi states that "it is still a mystery how PS1 mutations cause the disease" and wonders how some 150 different mutations scattered throughout the molecule can achieve the same result (AD). I would respond that missense mutations are known to subtly (or sometimes markedly) alter the native conformation of proteins. Because many of the PS1 mutations have been shown to increase Aβ42 generation, sometimes at the expense of Aβ40 generation, I think a highly plausible model is that their conformational effects increase the likelihood that the Aβ42-43 peptide bond within the APP transmembrane domain interacts efficiently with the two catalytic aspartates of presenilin. Indeed, the elegant FRET/FLIM analyses of Oksana Berezovska and colleagues support just such a PS1-APP conformational interaction model (6).

Dr. Marchesi's proposed feedback mechanism in which membrane-retained Aβ dimers might interact with PS1 and Aph-1 to destabilize the ?-complex itself seems to me to lack molecular specificity; that is, many other hydrophobic transmembrane fragments generated from γ-secretase substrates which have GxxxG motifs could do this, as well. Indeed, the more hydrophobic APP derivative, p3 (arising from a- and γ-secretase cleavages) would presumably be an even better candidate than Aβ for such an effect. While one can certainly not exclude such a feedback mechanism, I think it is biologically more plausible that Aβ- or p3-like substrate products of intramembranous cleavage are efficiently released into the luminal space of vesicles, where they might begin to oligomerize (due to very high local concentrations) and then be retained in part but also be secreted in part.

This brings me to the issue of the likelihood of retention of Aβ, whether in dimeric or monomeric form, within the membrane. Again, present information cannot exclude this occurring, at least in part. But the processing of a large and growing number of substrates by γ-secretase has led to the hypothesis that γ-secretase is conserved and ubiquitous because it efficiently removes single-transmembrane proteins from the bilayer. So, beyond its ability to release potential signaling domains from some of its many substrates (e.g., Notch), γ-secretase is viewed increasingly as a special type of "housekeeping" protease that serves to rid the membrane of otherwise long-lived transmembrane domains. If so, do the two resulting cleavage products get released quantitatively from the membrane? In healthy cultured cells expressing APP, one can detect abundant monomers of Aβ in the medium (as one can in plasma and CSF), and size-exclusion chromatography under non-denaturing conditions reveals that by far the major form of Aβ found in the medium is monomer [see, for example, Figure 2 in (7)]. Robust release of Aβ monomer following γ-secretase processing of endogenous APP has also been shown in primary human neurons (8). The CSF levels of secreted Aβ42 monomers (as measured by sandwich ELISAs that don't efficiently detect oligomers) are often significantly decreased in AD patients, even early in their clinical course, suggesting that extracellular monomer levels reflect the progressive accumulation of Aβ in myriad insoluble deposits in the cortex. These and other published data suggest to me that much of Aβ in the human brain occurs as soluble monomers (and small oligomers) that are released from cells. But again, such arguments cannot exclude a role for some Aβ peptides that remain in the membrane to contribute to the neuronal insult, whether as monomers or oligomers.

Regardless of one's perspective, Dr. Marchesi's intriguing hypothesis that membrane-retained Aβ dimers contribute to neuronal injury should be actively pursued experimentally. One way to do this might be to extend FRET/FLIM approaches to learn whether APP can be observed to undergo dimerization in living cells. For example, one might express two differentially tagged fluorescent APP molecules that can be temporally induced (e.g., via Tet-regulated constructs) in neuronal cells and then observe whether—and how quickly—they show FRET. Perhaps the same could be done to detect Aβ interactions within vesicular compartments of the cell or at the plasma membrane.

Finally, I conclude with a comment about the therapeutic implications of Dr. Marchesi's model. I don't concur with his interpretation that "many new therapies designed to reduce Aβ levels in AD patients are being proposed, but efforts to reduce peptide levels in blood and tissue spaces may not target the most toxic factors." Inhibitors of β- or γ-secretase currently under development are intended to lower both intracellular and cell-surface Aβ production (i.e., they are cell-penetrant), and they should be able to decrease the levels of intramembranous Aβ dimers. And immunotherapeutic approaches, which have shown salutary behavioral effects in AD mouse models and early evidence of clinical and neuropathological benefits in AD patients (9-11), could lower cerebral Aβ burden in a way that may secondarily affect intraneuronal levels, as well. Although specific anti-oligomer approaches are theoretically attractive, these may be more difficult to achieve than the foregoing examples now entering the clinic.

The very interesting review of Dr. Vincent Marchesi deals with the notion that Aβ, once generated by diverse secretases, never leaves the membrane and exerts its toxicity by yet unknown mechanisms. Supporting this view, we and others have seen evidence that intraneuronal accumulation of Aβ42 is the main trigger for the pathological events leading to neuron loss and brain atrophy. Increased Aβ42 has been observed in postmortem brains of patients with beginning Alzheimer disease and Down syndrome, and several APP transgenic mouse models.

Increasing intraneuronal Aβ42 leads to an age-dependent neuron loss in hippocampus in two different APP/PS1 transgenic mouse models with no correlation to plaque load, that is, extracellular Aβ. Intraneuronal Aβ42 is primarily found in multivesicular bodies in these mice. Intraneuronal Aβ42 accumulation in the somatodendritic compartment may have an influence on axonal trafficking and integrity, an issue which is currently also debated. However, whether Aβ42 is closely associated with intracellular membranes or plasma membrane in these mice is presently unclear.

Dr. Russo points out that if Aβ peptides or fragments of them remain within the lipid bilayer after their generation, Aβ42, with two additional hydrophobic residues, should be more stable within the bilayer than Aβ40, a factor that could account for its greater toxicity. He also raises the interesting possibility that the P-3 peptide, widely assumed to be nontoxic, whose sequence appears below, might be even more likely to partition within the bilayer, since it has the same hydrophobic core but lacks many of the polar residues of either Aβ fragment. If the β-secretase pathway is favored, as many suspect, there would be much less P-3 than either of the Aβs, but P-3 might be doing more than we realize. For one thing, its primary sequence exactly matches the homologous segments of the Aβs, as shown below, and it should, in principle, dimerize with either monomer. If the P-3 peptide is less toxic, for whatever reasons, could it function to neutralize the Aβs, acting as some kind of intramembranous chaperone? The effects of cholesterol on Aβ production are obviously complicated, as Russo points out, but the properties of lipid rafts and sterols must surely influence intramembranous transactions of the type I postulate.

Amino acid sequences of the Aβ42 peptide generated by the combined actions of β-secretase and γ-secretase, and the P-3 peptide generated by the combined actions of a-secretase and γ-secretase. P-3 is thought to be nontoxic.

Dr. Koo reminds us that the potentially toxic role of APP C-terminal fragments has a long history and is not consistent with the prevalent view that soluble Aβ aggregates are the major toxic principles. He wonders whether they represent a source of hydrophobic peptides of varying composition that might have unsuspected functions within the membrane interior. The idea that accumulations of C-terminal fragments might represent a reservoir of potentially toxic peptides is an intriguing speculation.

Dr. Selkoe raises interesting questions regarding sites where Aβ peptides accumulate inside neurons, and he proposes that a distinction should be made between Aβ production that might be "physiological" as opposed to that which is "pathological." He suggests that conditions in which Aβ is overexpressed (various transgenic models) might not reflect the sequence of events that takes place in the human condition that leads to AD, about which we have only fragmentary information. He believes that results from the study of brains of patients with trisomy 21 may be more revealing than the animal models, in that abundant amounts of extracellular Aβ are present at early ages in such patients. Very little Aβ is seen intracellularly, he contends, and that which is observed is believed to be due to the presence of Aβ dimers in secretory vesicles. There may indeed be important distinctions between the causes of dementia in Down syndrome and those which cause AD in non-trisomy individuals, but I don't think they contradict in a fundamental way the widely held view that Aβ peptides accumulate inside cells, both neuronal and non-neuronal, in the early stages of AD. Dr. Bayer's experience also supports this position.

Regarding the "mystery" as to why so many mutations of PS1 cause disease (150 as of now), I don't think it's wise, in this case, to fall back on the overused speculation that conformational changes are responsible. In the study Dr. Selkoe cites to support this idea (1), labeled antibodies to different proteins were used as donor and acceptor ligands. Because the fluorophores are attached to bulky, flexible macromolecules, and the analysis is done on fixed, antibody-stained cells, one can fairly question whether this approach has the resolving power to reliably detect conformational changes within individual protein molecules. FRET/FLIM analysis is used routinely to detect larger-scale interactions between neighboring proteins, but conclusions drawn from its use to study intramolecular conformations must be confirmed in other ways. So I believe the question of conformational influences is still open.

It is entirely reasonable to question how the interactions between hydrophobic peptides of similar sequences can be specific enough to account for the interactions I propose. There are now many examples where such interactions do indeed take place, so the ability to form stable associations is not in question. The GxxxG or GxxxA motifs that I propose to account, in part, for the ability of Aβ peptides to interact with other transmembrane proteins, if they are retained within the bilayer, are thought to be due to their ability to allow closer packing of neighboring polypeptides because they lack bulky side chains. I suspect that their ability to interact with specific proteins may be largely due to the fact that they are generated in close proximity to these potential partners. This is why I suggested that accumulations of such peptides, or fragments of them, might first interact with intact APP molecules or APH1, both of which are by definition at sites of Aβ generation. I have already noted, in response to the suggestion by Russo, that P-3 should also be one of these retained peptides. As suggested above, we should look into its fate more carefully than we have so far.

Another potential target/partner for Aβ is the neuronal sorting receptor sorLA, which appears to be involved in APP trafficking. A recent report (2) indicates that sorLA, a type-1 transmembrane protein of unknown function, colocalizes with APP in endosomal, Golgi, and plasma membranes, and may in some way block or reduce Aβ production. It is interesting that this receptor also has a transmembrane segment similar to the C-terminal end of Aβ42, as depicted below.

The C-terminal sequences of GVGFA (sorLA) and the GVVIA of Aβ overlap.

Furthermore, Dr. Selkoe cites an abundance of experimental evidence that Aβ peptides of various states of aggregation are found in the media of cultured cells, CSF, and other body fluids. I do not dispute these findings. However, they do not bear on whether or not a fraction of such peptides, or fragments of them, also exists in the interior of membranes for periods long enough to affect membrane function. Dr. Selkoe's proposal that the γ-secretase complex is a special type of "housekeeping" protease is itself an interesting speculation, and others in the field share a similar view (Kopan and Ilagan, 2004). I think it likely that such elaborate molecular machinery has evolved to generate peptides with specific biological functions, and we should figure out what they might be doing. It also seems reasonable to assume that since these peptides are derived from the intramembranous domains of transmembrane proteins, and are generated within the lipid bilayer, chances are good that they are also doing something biologically important within the membrane itself.

Dr. Marchesi provides refreshing insights into how accumulating membrane-associated Aβ may be involved in AD pathogenesis. For example, it is intriguing to consider that Aβ, which we thought accumulates in the inner aspect of outer limiting membranes of endosomes, may actually be embedded within the membrane bilayer. This could also illuminate why Aβ42 prefers to be retained in cells upon exocytosis of more soluble Aβ40 peptides.

Marchesi's Perspective article highlights the importance of investigating the biology of Aβ42 accumulating within neurons. What's the evidence for a detrimental role of intraneuronal Aβ, also in human AD? In short, by immuno-EM, accumulating intraneuronal Aβ42 and especially Aβ oligomers are associated with subcellular destruction, which equals neuronal dysfunction. This occurs in the absence of plaques. There is so much that needs to be uncovered, including how extracellular Aβ influences intracellular Aβ, the biology of APP/Aβ, PS1 and other AD-linked proteins, especially within synaptic membrane compartments, and the molecular mechanism by which accumulating intraneuronal Aβ initiates synaptic dysfunction. For a detailed review on intracellular Aβ biology, and a historical perspective, see Gouras et al., 2005.